D-allulose, a rare sugar and functional sweetener, can be biosynthesized by D-allulose 3-epimerase (DAEase). However, the the limited acid tolerance of the majority of reported DAEases severely limits their application. To develop a DAEase with enhanced catalytic activity and acid resistance for use under acidic conditions commonly encountered during juice processing, a phylogenetics-guided big-data mining and ancestral sequence reconstruction (ASR) strategy was employed to construct a novel DAEase (A3). Enzymatic characterization showed that the ancestral enzyme A3 retained more than 70% relative activity over a pH range of 4.0~7.0. After incubation at pH 4.0 for 6 h, A3 still retained 37.51% residual activity (10.09-fold higher than CcDAE), demonstrating enhanced catalytic performance and acid tolerance. Molecular dynamics simulation and structural analysis indicate that the presence of more hydrogen bonds, β4-α4 ring rigidity, and contraction of active-site tunnel entrances jointly resist acidic environments. Finally, the enzyme was applied to an acidic fruit juice system and successfully converted high-calorie sugars to D-allulose in situ, with conversion rates exceeding 30%. Collectively, these findings indicate that ancestral enzyme A3 is a novel DAEase that combines high catalytic performance and excellent stability, highlighting its potential for industrial applications. In summary, this study proposes a potential acid-resistant modification strategy for DAEase and provides an efficient, stable biocatalyst support for the industrial production of functional foods rich in D-allulose.